Modulation of the Proliferative Pathway, Neuroinflammation and Pain in Endometriosis

Endometriosis is a chronic disease characterized by pelvic inflammation. This study aimed at investigating the molecular mechanisms underlying the pathology and how they can be modulated by the administration of a natural compound, Actaea racemosa (AR). We employed an in vivo model of endometriosis in which rats were intraperitoneally injected with uterine fragments from donor animals. During the experiment, rats were monitored by abdominal high-frequency ultrasound analysis. AR was able to reduce the lesion’s size and histological morphology. From a molecular point of view, AR reduced hyperproliferation, as shown by Ki-67 and PCNA expression and MAPK phosphorylation. The impaired apoptosis pathway was also restored, as shown by the TUNEL assay and RT-PCR for Bax, Bcl-2, and Caspase levels. AR also has important antioxidant (reduced Nox expression, restored SOD activity and GSH levels, and reduced MPO activity and MDA levels) and anti-inflammatory (reduced cytokine levels) properties. Moreover, AR demonstrated its ability to reduce the pain-like behaviors associated with the pathology, the neuro-sensitizing mediators (c-FOS and NGF) expression, and the related central astrogliosis (GFAP expression in the spinal cord, brain cortex, and hippocampus). Overall, our data showed that AR was able to manage several pathways involved in endometriosis suppression.


Introduction
Endometrium-like stromal and glandular cells proliferate outside of the uterus in the persistent, excruciatingly painful illness known as endometriosis [1]. It is an inflammatory, estrogen-dependent condition that affects up to 40% of women undergoing fertility treatments and 7-11% of women during their reproductive years [2]. Patients' symptoms include severe dysmenorrhea, ongoing pelvic pain, and infertility [3]. Although several ideas have been published on the etiology of endometriosis, its pathophysiology is still unknown. The retrograde menstruation theory put forward by Sampson is the most frequently accepted [4]. This idea states that endometrial gland fragments are the source of endometriosis. During menstruation, retrograde migration allows unattached endometrial glands, cells, and debris to enter the peritoneum, where they can develop and implant. Hyperproliferation, poor apoptosis, and an inflammatory microenvironment all contribute to the formation of endometriotic lesions [5,6]. Therefore, cell death is crucial for maintaining homeostasis and eliminating surplus or unhealthy cells. A crucial defense mechanism against the development of endometriosis involves maintaining differentiated tissue through a delicate balance between cell proliferation and apoptosis. Recent research highlights the significance of the oxidative imbalance, inflammatory reactions, and associated persistent pain condition both at the lesion site and in the peritoneum [7,8].

Effect of AR Administration on Endometriotic Lesions Growth
Abdominal high-frequency ultrasound (hUS) analysis was employed to monitor the disease. Seven days after the endometriotis induction, no differences were detected between the groups ( Figure 1A, Endo group; Figure 1B, Endo + AR group) in lesion numbers ( Figure 1D, Endo group (2.8 ± 0.37), Endo + AR group (3 ± 0.32), p = 0.6938) and diameter ( Figure 1C, Endo group (0.22 ± 0.01), Endo + AR group (0.21 ± 0.01), p = 0.342). Seven days later, the analysis was repeated before the sacrifice. At this timepoint, the hUS exam showed an increased lesion diameter in the Endo group ( Figure 1E,G (0.74 ± 0.08), p = 0.0015) as compared to the animals treated with AR ( Figure 1F,G (0.43 ± 0.104), p = 0.0015). No difference between the groups was found in lesion number at this timepoint either ( Figure 1H, Endo group (3 ± 0.34), Endo + AR group (2.2 ± 0.58), p = 0.2610). Once the animals were sacrificed, a macroscopical analysis was conducted. Lesions harvested from the Endo group ( Figure 1I) showed higher area ( Figure 1K, 527.4 ± 42.50, p = 0.0002) and volume ( Figure 1L, 102.8 ± 2.4, p < 0.0001) as compared to the samples from the AR-administered rats ( Figure 1J,K, 168.8 ± 35.9, p = 0.0002; and Figure 1L The Kolmogorov-Smirnov test was applied to verify the normal distribution of the data, and then the t-test was applied. A p-value of less than 0.05 was considered significant. ** p < 0.01 vs. Endo; *** p < 0.001 vs. Endo. Endo + AR (J), lesion area (K), lesion volume (L). The Kolmogorov-Smirnov test was applied to verify the normal distribution of the data, and then the t-test was applied. A p-value of less than 0.05 was considered significant. ** p < 0.01 vs. Endo; *** p < 0.001 vs. Endo.

Discussion
Endometriosis is characterized by a proinflammatory and oxidative environment, hyperproliferation, and dysregulated apoptosis [29]. In this paper, we evaluated the molecular mechanisms of AR administration in endometriosis, focusing on proliferation, oxidative stress, and pain. The disease's development was monitored by hUS analysis. The administration of AR started once the pathology was established (first hUS analysis). Then, at the end of the experiment, the second hUS analysis showed a reduced lesion diameter, which was confirmed by the macroscopic analysis. AR reduced lesion volume and area. Additionally, there was a significant modification of the lesion histology, with a reduction of glands, stromal tissue, and fibrosis. From a molecular point of view, the smaller size of the lesions corresponds with a reduction in Ki-67 and PCNA expression. These two markers provide important information about cellular cell cycle dysregulation [30,31]. Ki-67 is involved in every phase of the cell cycle except for the G 1 phase, while PCNA is expressed in the phase of DNA synthesis only, and both are markers of proliferation [32]. AR administration significantly decreased the hyperproliferation that characterized the endometriotic lesions. Many intracellular signaling cascades stimulate cell proliferation. The molecular pathway involved in this stimulation is MAPK. This pathway is significantly disturbed in endometriosis and plays a key role in proliferative signaling. AR confirmed its anti-proliferative effect by reducing ERK and p38 phosphorylation. Hyperproliferation is accompanied by defective control of apoptosis [30]. Our molecular analysis confirmed the impaired expression of the anti-and pro-proteins and DNA fragmentation in the Endo group. AR administration restored the impaired apoptosis by Bax and Bcl-2 expression and the TUNEL assay. AR, apart from its antiproliferative effects, is known for its antioxidant properties. Excessive oxidative stress and depletion of antioxidants are closely associated with endometriosis [33]. Oxidative stress induces hyperproliferation of endometrial stroma, whereas antioxidants may limit stromal proliferation. Several studies have reported a significant decrease in the antioxidant defense, including SOD activity, and an increase in oxidized lipoproteins in the peritoneal microenvironment of women with endometriosis [34]. The increase in SOD activity was a result of oxidative stress, serving as an adaptive cellular response, accompanied by a decrease in GSH levels and an increase in MDA levels. [35]. Rats with endometriosis displayed an activation of the phagocytic cells in the innate immune system, as evidenced by the increased MPO activity in this inflammatory condition [36]. MPO is a critical enzyme of the innate immune system responsible for generating oxidant radicals. The antioxidant properties of AR restored the disturbed balance between oxidants and antioxidants in rats with endometriosis. This was demonstrated by the recovery of GSH levels, the reduction in SOD and MPO activity, and lipid peroxidation. AR administration also resulted in a reduction in the expression of Nox-1 and Nox-4, which are enzymes that play a crucial role in the synthesis of O 2 and H 2 O 2 . The experimental conditions revealed a close correlation between implant growth, the inflammatory microenvironment, and the manifestation of pain-related symptoms. As previously mentioned, the development of endometriosis is characterized by a significant increase in local inflammation and oxidative stress. This inflammation is observed to increase proportionally with the size of the cyst and the invasion of the peritoneal organs [37]. Recent evidence suggests that endometriosis worsens inflammatory symptoms and affects pain sensitivity [38]. Here, we examined the perception of pain in rats with endometriosis by conducting various tests that assess peripheral and visceral sensitivity. Consistent with previous research, our findings indicate that rats with endometriosis exhibit heightened visceral sensitivity. The animals that underwent endometriosis and were treated with AR showed decreased thermal and mechanical hyperalgesia and pain sensitivity. Endometriosis is associated with both central and peripheral sensitization, leading to increased vulnerability to pain [39]. First, tissue damage and inflammation sensitize the peripheral nociceptive system, causing a decrease in the pain threshold and an increase in the sensory input to the central nervous system. Persistent stimuli can lead to long-term changes in the central nervous system. This phenomenon is known as central sensitization, where the central response becomes disconnected from peripheral input [40]. Chronic pelvic pain and central sensitization can be induced by intensified painful stimuli. The hippocampus is considered one of the key brain regions involved in the emotional and cognitive consequences of neuropathic pain. Abnormal connectivity in the hippocampus and afferences to the frontoinsular and somatosensory cortex were observed in patients with endometriosis [41]. These particular regions of the brain are associated with the shift from short-term, acute pain to long-term, chronic pain [42,43]. AR administration prevented astrogliosis in the spinal cord and hippocampal tissue. Indeed, it strongly reduced the expression of neuroinflammatory mediators.

Experimental Protocol
The rats were allocated randomly to two groups, one being donors and the other recipients, and endometriosis was induced in accordance with the previously outlined method [44]. To ensure uniform estrogen levels in the rats, the donor animals were given a dose of 10 IU pregnant mare serum gonadotropin. At the 41 h mark, the rats were euthanized, and their uteri were excised. The tissue was finely chopped using scissors and placed in a centrifuge tube of 1.5 mL capacity that contained PBS. The tissue from all the donor rats was combined, and an amount equivalent to one uterus per 500 µL of PBS was administered via intraperitoneal injection along the midventral line of the recipient rats. A period of seven days was allotted for the development of endometriosis. A success rate of 70% was observed for the development of the lesions [45].

Experimental Groups
The rats were allocated randomly and grouped as follows (N = 35 per group): (1) Endo group: experimental endometriosis was induced in the rats, and they were orally administered with vehicle (saline) using a gavage on the seventh day and subsequently for the following seven days; (2) Endo + AR group: experimental endometriosis was induced in the rats, and they were orally administered with AR (100 mg/Kg) using a gavage on the seventh day and subsequently for the following seven days; (3) Control group: the rats were given an intraperitoneal injection of 500 µL of PBS instead of endometrial tissue, and they received a vehicle (saline) via oral gavage on the seventh day and for the subsequent seven days.
The AR dose was based on previous studies [26]. To assess the impact of administering AR on endometriotic-like lesions, the rats were euthanized 14 days after their induction. Subsequently, a laparotomy was conducted to retrieve the endometriotic implants for additional analyses (Figure 7).

Abdominal High-Frequency Ultrasound
Pelvic ultrasound was performed to monitor the development of the endometriotic lesions at seven and fourteen days after the implant. The analysis included the anterior and posterior pelvic areas to reach the lesions in both locations. Ultrasonographic exams were performed by the Esaote MYLAB OMEGA (Esaote Italia, Milan, Italy) on anesthetized rats (2% isoflurane) positioned in dorsal recumbency. Abdominal B-mode was performed with a high-frequency linear array (4-15 MHz) transducer. Longitudinal and transverse scanning planes were employed for the evaluation of different abdominal structures [46]. All analyses were performed double-blind.

Behavioral Analysis
Behavioral analyses were performed 14 days after the endo induction.

Open Field Test
The measurement of locomotor activity and exploratory behavior was carried out using a square open-field arena [47]. Following a one-minute habituation period, each rat was positioned in one corner of the arena and monitored for five minutes. A 20% ethanol solution was utilized to clean the equipment after each analysis. The recorded parameters included the number of times animals crossed with four legs (spontaneous locomotion), entries into the central square, and time spent in the central square (in seconds).

Hot Plate
The hot plate test was employed to assess the pain threshold to thermal stimuli [48]. The rats were permitted to walk on a hot plate (at a temperature of 53.0 ± 0.1 °C) for a maximum duration of 45 s.

Elevated Plus Maze Test
The apparatus for the elevated plus maze consisted of two enclosed arms and two open arms, which were connected via a central square [49]. The rat was placed in the apparatus and allowed to move around freely for 5 min. A solution containing 20% ethanol was used to clean the apparatus after each analysis.

Acetic-Acid-Induced Abdominal Contractions
The animals were administered an intraperitoneal injection of 0.6% acetic acid, and the number of writhes induced by the acid was observed for 20 min, starting 5 min after the administration [50]. The stretching of the hind limbs followed by a contraction of the abdomen was defined as a writhe.

Abdominal High-Frequency Ultrasound
Pelvic ultrasound was performed to monitor the development of the endometriotic lesions at seven and fourteen days after the implant. The analysis included the anterior and posterior pelvic areas to reach the lesions in both locations. Ultrasonographic exams were performed by the Esaote MYLAB OMEGA (Esaote Italia, Milan, Italy) on anesthetized rats (2% isoflurane) positioned in dorsal recumbency. Abdominal B-mode was performed with a high-frequency linear array (4-15 MHz) transducer. Longitudinal and transverse scanning planes were employed for the evaluation of different abdominal structures [46]. All analyses were performed double-blind.

Behavioral Analysis
Behavioral analyses were performed 14 days after the endo induction.

Open Field Test
The measurement of locomotor activity and exploratory behavior was carried out using a square open-field arena [47]. Following a one-minute habituation period, each rat was positioned in one corner of the arena and monitored for five minutes. A 20% ethanol solution was utilized to clean the equipment after each analysis. The recorded parameters included the number of times animals crossed with four legs (spontaneous locomotion), entries into the central square, and time spent in the central square (in seconds).

Hot Plate
The hot plate test was employed to assess the pain threshold to thermal stimuli [48]. The rats were permitted to walk on a hot plate (at a temperature of 53.0 ± 0.1 • C) for a maximum duration of 45 s.

Elevated Plus Maze Test
The apparatus for the elevated plus maze consisted of two enclosed arms and two open arms, which were connected via a central square [49]. The rat was placed in the apparatus and allowed to move around freely for 5 min. A solution containing 20% ethanol was used to clean the apparatus after each analysis.

Acetic-Acid-Induced Abdominal Contractions
The animals were administered an intraperitoneal injection of 0.6% acetic acid, and the number of writhes induced by the acid was observed for 20 min, starting 5 min after the administration [50]. The stretching of the hind limbs followed by a contraction of the abdomen was defined as a writhe.

Abdominal High-Frequency Ultrasound
An ultrasonographic examination was conducted on anesthetized rats (2% isoflurane) placed in dorsal recumbency, using the Esaote MYLAB OMEGA VET. A B-mode ultrasound of the abdomen was carried out using a high-frequency linear array transducer (4-15 MHz) [46]. Both longitudinal and transverse scanning planes were utilized to examine various abdominal structures.

Histological Examination
The endometriotic lesions were fixed in a formaldehyde solution and then embedded in Paraplast [51]. Tissue slides were stained with H&E and evaluated using a Leica DM6 microscope (Leica Microsystems SpA, Milan, Italy). A histological analysis was performed using a double-blind procedure. Histopathological scores were assigned according to the formula P (persistence of epithelial cells in the explants) × I (intensity of glands), as already described [52]. The lesion volume was calculated according to the formula V = (length × width 2 ) × 0.5 [53]. Lesions fibrosis was evaluated by Masson trichrome staining (Bio-Optica, Milan, Italy) [54].

Terminal Deoxynucleotidyl Nick-End Labeling (TUNEL) Assay
Apoptosis was analyzed with a TUNEL assay using an in situ cell death detection kit (Roche 11684795910) [55].

Biochemical Analysis
The TBARS test was used to assess lipid peroxidation by measuring MDA levels at 535 nm [61]. SOD activity was evaluated as already described [62] and is expressed as U/g protein [63]. GSH levels were determined using a microplate reader at 412 nm [64].

RNA Extraction and cDNA Synthesis
An RNeasy kit (Qiagen, Milan, Italy) was employed to extract RNA for real-time polymerase chain reaction (RT-PCR) analysis. Quantification was performed on RNA with a spectrophotometer (NanoDrop Lite). An iScript RT-PCR kit (Bio-Rad, Hercules, CA, USA) was used to synthesize first-strand cDNA [65].

Immunohistochemical Analysis
Immunohistochemical localization of anti-GFAP (Proteintech, catalog number 16825-1-AP, dilution 1:5000) was performed in the spinal cord, brain cortex, and hippocampus as already described [67]. All sections were incubated with the primary antibody, then washed with PBS and treated as previously reported [68]. Stained sections were observed using a Leica DM6 microscope (Leica Microsystems SpA, Milan, Italy).

Statistical Analysis
The Kolmogorov-Smirnov test was applied to verify the normal distribution of the data, and then the t-test was applied when comparing the two groups (Prism 8 for macOS version 8. 2.1 (279)). In the analyses with three groups, the results were analyzed by one-way ANOVA followed by a Bonferroni post hoc test for multiple comparisons. A p-value of less than 0.05 was considered significant. # p < 0.05 vs. CTL, ## p < 0.01 vs. CTL, ### p < 0.001 vs. CTL, * p < 0.05 vs. Endo, ** p < 0.01 vs. Endo, *** p < 0.001 vs. Endo.

Conclusions
This paper focused on the molecular mechanisms involved in AR administration during endometriosis. AR administration strongly reduced the pathology's development and the lesion size, showing anti-proliferative and pro-apoptotic effects. The data collected highlighted the AR impact on enhancing the activity of ROS-scavenging enzymes (SOD) and endogenous antioxidant systems (GSH), while suppressing the activity of ROS producing enzymes (Nox). We observed a significant correlation between the inflammatory microenvironment, the growth of endometrial implants, and the development of pain-like symptoms under our experimental conditions. AR administration reduced the expression of neuro-sensitizing mediators, which, in turn, led to reduced activation of astrocytes in the spinal cord, cortex, and hippocampus.  Data Availability Statement: Based upon the rules of our laboratory, the datasets used in the current study are available from the corresponding author on reasonable request.

Conflicts of Interest:
The authors declare no conflict of interest.